Stochastic state transitions give rise to phenotypic equilibrium in populations of cancer cells

Cancer cells within individual tumors often exist in distinct phenotypic states that differ in functional attributes. While cancer cell populations typically display distinctive equilibria in the proportion of cells in various states, the mechanisms by which this occurs are poorly understood. Here, we study the dynamics of phenotypic proportions in human breast cancer cell lines. We show that subpopulations of cells purified for a given phenotypic state return towards equilibrium proportions over time. These observations can be explained by a Markov model in which cells transition stochastically between states. A prediction of this model is that, given certain conditions, any subpopulation of cells will return to equilibrium phenotypic proportions over time. A second prediction is that breast cancer stem-like cells arise de novo from non-stem-like cells. These findings contribute to our understanding of cancer heterogeneity and reveal how stochasticity in single-cell behaviors promotes phenotypic equilibrium in populations of cancer cells.     

Molecular architecture and connectivity of the budding yeast Mtw1 kinetochore complex

Kinetochores are large multiprotein complexes that connect centromeres to spindle microtubules in all eukaryotes. Among the biochemically distinct kinetochore complexes, the conserved four-protein Mtw1 complex is a central part of the kinetochore in all organisms. Here we present the biochemical reconstitution and characterization of the budding yeast Mtw1 complex. Direct visualization by electron microscopy revealed an elongated bilobed structure with a 25-nm-long axis. The complex can be assembled from two stable heterodimers consisting of Mtw1p-Nnf1p and Dsn1p-Nsl1p, and it interacts directly with the microtubule-binding Ndc80 kinetochore complex via the centromere-proximal Spc24/Spc25 head domain. In addition, we have reconstituted a partial Ctf19 complex and show that it directly associates with the Mtw1 complex in vitro. Ndc80 and Ctf19 complexes do not compete for binding to the Mtw1 complex, suggesting that Mtw1 can bridge the microtubule-binding components of the kinetochore to the inner centromere.     

Cohesin mediates chromatin interactions that regulate mammalian β-globin expression

The β-globin locus undergoes dynamic chromatin interaction changes in differentiating erythroid cells that are thought to be important for proper globin gene expression. However, the underlying mechanisms are unclear. The CCCTC-binding factor, CTCF, binds to the insulator elements at the 5' and 3' boundaries of the locus, but these sites were shown to be dispensable for globin gene activation. We found that, upon induction of differentiation, cohesin and the cohesin loading factor Nipped-B-like (Nipbl) bind to the locus control region (LCR) at the CTCF insulator and distal enhancer regions as well as at the specific target globin gene that undergoes activation upon differentiation. Nipbl-dependent cohesin binding is critical for long-range chromatin interactions, both between the CTCF insulator elements and between the LCR distal enhancer and the target gene. We show that the latter interaction is important for globin gene expression in vivo and in vitro. Furthermore, the results indicate that such cohesin-mediated chromatin interactions associated with gene regulation are sensitive to the partial reduction of Nipbl caused by heterozygous mutation. This provides the first direct evidence that Nipbl haploinsufficiency affects cohesin-mediated chromatin interactions and gene expression. Our results reveal that dynamic Nipbl/cohesin binding is critical for developmental chromatin organization and the gene activation function of the LCR in mammalian cells.     

Contribution of the Ly49E Natural Killer Receptor in the Immune Response to Plasmodium berghei Infection and Control of Hepatic Parasite Development

Natural killer (NK) cells have different roles in the host response against Plasmodium-induced malaria depending on the stage of infection. Liver NK cells have a protective role during the initial hepatic stage of infection by production of the T_H1-type cytokines IFN-γ and TNF-α. In the subsequent erythrocytic stage of infection, NK cells also induce protection through Th1-type cytokines but, in addition, may also promote development of cerebral malaria via CXCR3-induction on CD8⁺ T cells resulting in migration of these cells to the brain. We have recently shown that the regulatory Ly49E NK receptor is expressed on liver NK cells in particular. The main objective of this study was therefore to examine the role of Ly49E expression in the immune response upon Plasmodium berghei ANKA infection, for which we compared wild type (WT) to Ly49E knockout (KO) mice. We show that the parasitemia was higher at the early stage, i.e. at days 6–7 of Plasmodium berghei ANKA infection in Ly49E KO mice, which correlated with lower induction of CD69, IFN-γ and TNF-α in DX5⁻ liver NK cells at day 5 post-infection. At later stages, these differences faded. There was also no difference in the kinetics and the percentage of cerebral malaria development and in lymphocyte CXCR3 expression in WT versus Ly49E KO mice. Collectively, we show that the immune response against Plasmodium berghei ANKA infection is not drastically affected in Ly49E KO mice. Although NK cells play a crucial role in Plasmodium infection and Ly49E is highly expressed on liver NK cells, the Ly49E NK receptor only has a temporarily role in the immune control of this parasite.     

Current Practice of Public Involvement Activities in Biomedical Research and Innovation: A Systematic Qualitative Review

Background

A recent report from the British Nuffield Council on Bioethics associated ‘emerging biotechnologies’ with a threefold challenge: 1) uncertainty about outcomes, 2) diverse public views on the values and implications attached to biotechnologies and 3) the possibility of creating radical changes regarding societal relations and practices. To address these challenges, leading international institutions stress the need for public involvement activities (PIAs). The objective of this study was to assess the state of PIA reports in the field of biomedical research.

Methods

PIA reports were identified via a systematic literature search. Thematic text analysis was employed for data extraction.

Results

After filtering, 35 public consultation and 11 public participation studies were included in this review. Analysis and synthesis of all 46 PIA studies resulted in 6 distinguishable PIA objectives and 37 corresponding PIA methods. Reports of outcome translation and PIA evaluation were found in 9 and 10 studies respectively (20% and 22%). The paper presents qualitative details.

Discussion

The state of PIAs on biomedical research and innovation is characterized by a broad range of methods and awkward variation in the wording of objectives. Better comparability of PIAs might improve the translation of PIA findings into further policy development. PIA-specific reporting guidelines would help in this regard. The modest level of translation efforts is another pointer to the “deliberation to policy gap”. The results of this review could inform the design of new PIAs and future efforts to improve PIA comparability and outcome translation.     

Reconstruction of a windborne insect invasion using a particle dispersal model,historical wind data,and Bayesian analysis of genetic data

Understanding how invasive species establish and spread is vital for developing effective management strategies for invaded areas and identifying new areas where the risk of invasion is highest. We investigated the explanatory power of dispersal histories reconstructed based on local‐scale wind data and a regional‐scale wind‐dispersed particle trajectory model for the invasive seed chalcid wasp Megastigmus schimitscheki (Hymenoptera: Torymidae) in France. The explanatory power was tested by: (1) survival analysis of empirical data on M. schimitscheki presence, absence and year of arrival at 52 stands of the wasp's obligate hosts, Cedrus (true cedar trees); and (2) Approximate Bayesian analysis of M. schimitscheki genetic data using a coalescence model. The Bayesian demographic modeling and traditional population genetic analysis suggested that initial invasion across the range was the result of long‐distance dispersal from the longest established sites. The survival analyses of the windborne expansion patterns derived from a particle dispersal model indicated that there was an informative correlation between the M. schimitscheki presence/absence data from the annual surveys and the scenarios based on regional‐scale wind data. These three very different analyses produced highly congruent results supporting our proposal that wind is the most probable vector for passive long‐distance dispersal of this invasive seed wasp. This result confirms that long‐distance dispersal from introduction areas is a likely driver of secondary expansion of alien invasive species. Based on our results, management programs for this and other windborne invasive species may consider (1) focusing effort at the longest established sites and (2) monitoring outlying populations remains critically important due to their influence on rates of spread. We also suggest that there is a distinct need for new analysis methods that have the capacity to combine empirical spatiotemporal field data, genetic data, and environmental data to investigate dispersal and invasion.     

Genome Editing by CRISPR/Cas9: A Game Change in the Genetic Manipulation of Protists

Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR‐associated gene 9) system has been transformative in biology. Originally discovered as an adaptive prokaryotic immune system, CRISPR/Cas9 has been repurposed for genome editing in a broad range of model organisms, from yeast to mammalian cells. Protist parasites are unicellular organisms producing important human diseases that affect millions of people around the world. For many of these diseases, such as malaria, Chagas disease, leishmaniasis and cryptosporidiosis, there are no effective treatments or vaccines available. The recent adaptation of the CRISPR/Cas9 technology to several protist models will be playing a key role in the functional study of their proteins, in the characterization of their metabolic pathways, and in the understanding of their biology, and will facilitate the search for new chemotherapeutic targets. In this work we review recent studies where the CRISPR/Cas9 system was adapted to protist parasites, particularly to Apicomplexans and trypanosomatids, emphasizing the different molecular strategies used for genome editing of each organism, as well as their advantages. We also discuss the potential usefulness of this technology in the green alga Chlamydomonas reinhardtii.